Abstract

The activation of oocytes is one of the most important steps for a successful cloning, and chemicals used for activation can affect the viability of cloned offspring. Therefore, some of them may be omitted for activation to eliminate their possible detrimental effect on nuclear transfer (NT) embryos. The objective of this study was to examine the effect of calcium ionophore (CaI, A23187, Sigma, St. Louis, MO, USA) and cytochalasin D (CD) on activation and in vitro development of nuclear transfer units derived from bovine granulosa cells (GCs) treated with the cell cycle inhibitor, roscovitine. Bovine oocytes isolated from slaughterhouse ovaries were matured in TCM199 supplemented with fetal bovine serum (FBS), sodium pyruvate, penicillin/streptomycin, rIGF-1, bFSH, and bLH. GCs were isolated from ovarian follicles and cultured in DMEM-F12 supplemented with 10% FBS at 37°C in 5% CO2 in air. Prior to NT, donor cells were exposed to 15 mM roscovitine for 24 hours and small cells were used for NT. A single cell was inserted into the perivitelline space of the enucleated oocyte. Oocyte-cell couples were fused using a 20 μs DC pulse of 40V/150 μm. Two hours after fusion, NT units were assigned into four groups and activated by CaI (5 μM for 10 min.), and then incubated with cycloheximide (CHX, 10 μg mL-1) + CD (2.5 μg mL-1) for 1 h, followed by CHX alone (without CD) for 5 h (Group I) or CaI (for 10 min.), followed by CHX alone for 6 h (Group II). In the Group III, NT units were activated by CHX (10 μg mL-1) + CD (2.5 μg mL-1) for 1 h, followed by CHX for 5 h, and Group IV, CHX alone for 6 h. The base activation medium was TCM199 with 1% FBS for CaI and 10% FBS for CHX and CD. After activation, NT units were cultured for 7 days in BARC medium. Differences in activation, cleavage and blastocyst formation rates among treatments were analyzed by one-way ANOVA after arcsin square transformation. The results are summarized in Table 1. Our data showed that CaI and CD did not affect the activation and in vitro development of NT embryos derived from roscovitine-treated GCs. It suggests that both chemicals may be redundant during cloning procedure. This study was supported by a grant from ProLinia, Inc and TUBITAK, Turkey (VHAG-1908-102V048).